Background & Aims: Recent studies have emphasized causative links between microRNA (miRNA) deregulations and cancer development. In hepatocellular carcinoma (HCC), information on differentially expressed miRNA remained largely undefined. Methods: Array-based miRNA profiling was performed on HCC cells that were derived from chronic carriers of hepatitis B virus (HBV) and hepatitis C virus (HCV), and nonviral-associated patients. Specific microRNA (miR)-223 and miR-222 deregulations were verified in an independent series of tumors. The functional effect of miR-223 was examined further. An integrative analysis of messenger RNA (mRNA) array with in silico predictions defined potential downstream targets of miR-223. A luciferase reporter assay was conducted to confirm target association. Results: Distinct up-regulations of miR-222, miR-221, and miR-31, and down-regulations of miR-223, miR-126, and miR-122a were identified. Further investigations suggested the highly deregulated miR-223 and miR-222 could unequivocally distinguish HCC from adjacent nontumoral liver, irrespective of viral associations (P ≤ .0002). Re-expression of miR-223 in HBV, HCV, and non-HBV non-HCV–related HCC cell lines revealed a consistent inhibitory effect on cell viability (P < .01). Integrative analysis further implicated Stathmin 1 (STMN1) as a downstream target of miR-223. A strong inverse relationship between STMN1 mRNA and miR-223 expressions was shown (P = .006). A substantial reduction in STMN1 protein was further demonstrated upon restoration of miR-223 expression in HCC cell lines. We further showed that miR-223 readily could suppress the luciferase activity in reporter construct containing the STMN1 3' untranslated region (P = .02). Conclusions: Our study revealed specific miRNA differential expressions in HCC and underscores the potential importance of miR-223 down-regulations in the development of HCC.